Immunization of Basa fish (Pangasius bocourti) against ...jifro.ir/article-1-3622-fa.pdf · immunization of Basa fish against Ich with live and sonicated trophonts by intraperitoneal

Iranian Journal of Fisheries Sciences 17(4) 763-775 2018

DOI:10.22092/ijfs.2018.119247

Immunization of Basa fish (Pangasius bocourti) against

Ichthyophthirius multifiliis with live and sonicated trophonts

Uawonggul N.1*

; Rattanamalee C.1; Daduang S.

2

Received: September 2016 Accepted: April 2017

Abstract

The high density of Basa fish (Pangasius bocourti) culture leads to outbreaks of

Ichthyophthirius multifiliis (Ich), also knows as white spot disease. In this research,

immunization of Basa fish against Ich with live and sonicated trophonts by

intraperitoneal (IP) injection was investigated. Anti-Ich antibody titer was determined

using ELISA and Western immunoblotting 21 days post immunization. The results

revealed that pre-immunized fish, non-immunized fish and fish immunized with bovine

serum albumin (BSA) at a concentration 65 µg g-1

fish did not show specific antibody

against Ich. 21 days post immunization, fish immunized with live trophonts exhibited

higher anti-Ich antibody titer than fish immunized with sonicated trophonts at the same

antigen concentration. Fish immunized with 65 µg trophonts protein/g fish live

trophonts showed the highest titer 1:1,000 (p<0.05). The results from Western

immunoblotting showed two parasite protein bands of 66 kDa and <14 kDa, which

reacted with antibodies from serum of immune fish. No fish in the non-immunized

group survived. At the same concentration of antigen (65 µg g-1

), fish immunized with

live trophonts exhibited the highest survival rate, 63.33±5.77% (p<0.05). Therefore,

these results are the Basa fish immunizing procedure will be the way to conduct

immunization against Ich to prevent disease outbreaks in aquaculture.

Keywords: Pangasius bocourti, Ichthyophthirius multifiliis, Immunization

1-Faculty of Science, Nakhon Phanom University, Nakhon Phanom, 48000, Thailand

2-Department of Pharmacology and toxicology, Faculty of Pharmaceutical Sciences,

Khon Kaen University, Khon Kaen, 40002, Thailand

*Corresponding author's Email: [email protected]

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http://jifro.ir/article-1-3622-fa.html

764 Uawonggul et al., Immunization of Basa fish against Ichthyophthirius multifiliis with…

Introduction

Basa fish or Asian catfish (Pangasius

bocourti) is a freshwater fish in the

family Pangasiidae found in Southeast

Asia, especially in the Mekong and

Chao Phraya Rivers. This fish has an

important economic impact in Nakhon

Phanom Province, Thailand. It is

cultured in cages in the Mekong basin

and exported to the other Southeast

Asian countries as well as to Europe

and Russia. It is prized for its white

meat, tender texture, good taste, low fat

and easily digestible protein (Hung et

al., 2002; Jiwyam, 2010; Sriphairoj et

al., 2010). Increasing consumer demand

has encouraged rapid extension of Basa

fish farming. This form of aquaculture

involves high density fish culture which

unfortunately has led to disease

outbreaks (Dickerson and Findly,

2014). Antibiotics as oxytetracycline

are used to control and treat this

disease. Their dosages are usually high

which promotes antibiotic resistance in

microorganisms, low meat quality and

environmental pollution (Saini et al.,

2014). White spot disease is caused by

Ichthyophthirius multifiliis (Ich) and is

commonly found in native and farmed

freshwater fish. Diseased native fish

can spread Ich to farmed fish (Xu et al.,

2013). Many infections have been

reported in fish cultures, especially in

tropical and subtropical regions

(Scholz, 1999). Ich can spread rapidly

in poor quality water or unsuitable

culture conditions (Osman et al., 2009).

In Ich infected fish skin and gills are

damaged leading to fish mortality,

especially in fingerlings. This

represents a significant economic loss

in aquaculture (Martins et al., 2011).

Currently, chemicals such as formalin,

methylene blue, copper sulfate,

potassium permanganate, hydrogen

peroxide and acetic acid are used to

treat fish against this parasite (Xiao-

Feng et al., 2014; Song et al., 2015).

The most effective chemical used for

treatment is malachite green, which was

banned due to its toxicity (Song et al.,

2015). The chemicals presently used are

applied for treatment only after the

presence of parasite has been detected

on fish skin. Thus, the treatment is not

effective enough (Xu et al., 2008a, b).

Moreover, Ich treatment is more

complicated due to its life cycle. This

parasite has a temperature dependent

life cycle that includes three stages,

theront, trophont and tomont (Shinn et

al., 2009). Tomont referred as cyst of

Ich in a free form living in an aquatic

environment. Cyst walls block chemical

penetration making treatment difficult.

Infected fish may acquire systemic and

mucosal immunity against Ich

(Swennes et al., 2007; Alvarez-

Pellitero, 2008). Fish vaccination is an

alternative choice for parasite

prevention that is harmless to the

environment. Several studies reported

successful fish immunization against

Ich. Dalgaard et al. (2002) reported

protection against Ich in rainbow trout

(Oncorhynchus mykiss) after

immunization with sonicated formalin

killed trophonts. Heidarieh et al. (2014)

reported an immune response against

Ich in rainbow trout immunized with

irradiated trophonts. Xu et al. (2008a)

reported immunization of Nile tilapia

(Oreochromis niloticus) against Ich by

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Iranian Journal of Fisheries Sciences 17(4) 2018 765

immersing them in water containing

live theronts and sonicated trophonts as

well as by intraperitoneal (IP) injection

at levels of 20,000 theronts per fish and

65 µg trophont protein g-1

fish,

respectively. The results showed that

anti-Ich antibody of fish immunized by

immersion in water containing live

theronts and by IP injection of live

theronts and sonicated trophonts had

significantly higher anti-Ich antibodies

than fish immunized with sonicated

trophonts by immersion and non-

immunized fish. Moreover, Xu et al.

(2008b) reported that the channel

catfish immunized with formalin killed

and freeze-thawed trophonts did not

produced anti-Ich antibodies and were

infected by Ich. Osman et al. (2009)

immunized Goldfish (Carassius

auratus) against Ich at levels 20,000

live theronts per fish, 2,000 trophonts

per fish and 65 µg sonicated trophonts/

g fish by immersion and IP injection.

Fish immunized with theronts by

immersion and IP injection showed

higher anti-Ich antibodies and survival

rates than fish immunized with

trophonts by immersion and IP

injection. Martins et al. (2011)

evaluated temperature effects on

Ictalurus punctatus immune response

against live theronts of Ich and found

that immunized fish at low temperature

had high mortality and no anti-Ich

antibody production. However,

immunity of Basa fish against Ich is not

clear. Therefore, this study aims to

investigate immune response of Basa

fish against Ich after immunization with

live and sonicated trophonts.

Materials and methods

Fish, parasite and water quality

Basa fish, having a mean length of

8.9±0.7 cm mean, mean weight of

11.30±1.93 g and no previous infection

with Ich were cultured in glass tanks at

a density of 30 fish 100 L-1

of

dechlorinated tap water. Fish were

distributed among 8 treatments with 2

replicates and were fed 40% crude

protein at a level of 5% of their body

weight daily using commercial fish

feed. Infected Basa fish were obtained

from Nakhon Phanom Inland Fisheries

Research and Development Center.

These infected fish were rinsed with

distilled water and fish skin was gently

scraped to isolate Ich. Water quality

measurement and water exchange were

performed every three days. Water

temperature, pH and dissolve oxygen

(DO) were determined using a pH

meter (HI-98127, Hanna, UK) and DO

meter (HI9147, Hanna, UK),

respectively. Ammonia and nitrite

concentrations were measured using

ammonia and nitrite test kits (Advance

Pharma, Thailand). During the

experiment, water temperature, pH,

DO, ammonia and nitrite concentrations

were 31.3±2.4 C, 7.13±0.30, 6.8±0.5

mg L-1

, 0.2±0.1 mg L-1

, 0.2±0.2 mg L-1

,

respectively.

Antigen preparation

Antigen preparation was modified from

Xu et al. (2008a). Trophonts were

separated from fish skin and mucus

then pooled in a plastic tube. The

collected trophonts were counted with

Sedgewick Rafter counting chamber

(SPI Supplies, USA) and concentrated

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by centrifugation at 1,000 rpm for 5

min. Then the supernatant was

discarded and the pellet resuspended in

PBS buffer, pH 7.4. The trophont

suspension was used as the first antigen

(live trophonts). Another antigen

(sonicated trophonts) was prepared by

sonicating a trophont suspension for 1

min on ice. The protein concentration in

live and sonicated trophonts was

measured using a spectrophotometric

method (HALO RB-10, Dynamica,

Australia) (Bradford, 1976).

Immunization

Fish were divided into 8 treatments and

immunized by IP injection with 100 µL

of antigen. Antigen was mixed with

Freund’s adjuvant before injection. The

first treatment was non-immunized fish.

The second treatment was immunized

with bovine serum albumin (BSA) at 65

µg protein g-1

fish. First and second

treatments were used as negative

controls. Fish in treatments 3-5 were

immunized with live trophonts at 45, 55

and 65 µg protein g-1

fish, while fish in

treatments 6-8 were immunized with

sonicated trophonts at same respective

protein concentrations.

Serum sampling

Before immunization, fish serum was

kept as pre-immunized. Twenty one

days post immunization, five fish in

each of treatments 1-8 were randomly

selected for serum sampling. Fish blood

was sampled from the base of the tail.

Blood coagulation was allowed at 4 C

overnight and then the samples were

centrifuged at 10,000 rpm at 4 C for 10

min (Z236K, Hermle, Germany). The

antibody titer against Ich in the

collected serum was determined using

ELISA and Western immune blotting.

ELISA

ELISA was performed according to

Kuendee et al. (2015) with slight

modification. Ich was diluted in a

carbonate buffer, pH 9.5 to 20 µg mL-1

.

Each well of 96-well polystyrene plates

(Nunc, Denmark) was coated with 50

µL of the diluted Ich solution. The

coated plate was washed 3 times with

TBST and blocked with 5% skim milk

(Scharalau, Spain). Then, the wells

were added with various serial dilutions

of fish serum and incubated at 37 C for

1 hr. After that, it was washed 3 times

with TBST and incubated with mouse

anti-Basa fish IgM antiserum (1:105) at

37 C for 1 hr, followed by washing 3

times with TBST again. Afterward, the

plate was incubated with conjugated

anti-mouse IgG linked with alkaline

phosphatase (1:5103, Zymed, USA) at

37 C for 1 hr. Subsequently, it was

washed 3 times with TBST and 3 times

with TBS. 100 µL of 1 mg mL-1

-

Nitrophenyl phosphate (Amersham,

Canada) was added as substrate. Next,

the absorbance was determined at 405

nm using a Microplate Reader (Bio-

Rad, USA). The titer in this study was

defined as the highest dilution that still

showed a positive result.

Western immunoblotting

Ich proteins were separated with 12%

separing gel and 4% stacking gel with

constant voltage at 150 V. Then, the

proteins were transferred from SDS-

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PAGE gel onto a nitrocellulose

membrane. After this transfer, the

membrane was divided as a part was

allocated for protein molecular weight

estimation and a part of the membrane

was used for samples immunoblotting.

The marker part of the membrane part

stained with 0.1% Amido Black,

whereas the sample part of the

membrane was incubated in 5% skim

milk. After incubation, the sample part

of the membrane was further incubated

with a various serial dilution of fish

serum as 1:10, 1:102, 1:10

3, 1:10

4, 1:10

5

and 1:106 at room temperature for1 hr

and washed 3 times with TBST. Next, it

was incubated with anti-Basa fish

serum (1: 104) at room temperature for1

hr. After that, the membrane was

washed 3 times with TBST and

incubated with anti-mouse IgG linked

with alkaline phosphatase (1: 5103) at

room temperature for1 hr.

Subsequently, it was washed 3 times

with TBST and 3 times with TBS.

Then, the membrane was soaked twice

in a substrate buffer, pH 9.5 and added

with a NBT/BCIP substrate solution

(Bio-Rad, USA). Finally, this

membrane was agitated in a substrate

solution until the protein band could be

observed.

Challenge with Ich

Twenty one days post immunization, 10

fish from each group were transferred

to new glass tank containing with 100 L

of dechlorinated tap water. These fish

were challenged with 104 theronts fish

-1

for 2 hr. White spots on the fish skin

and scratching behavior against the

walls of the tank were observed and

recorded. Two days post challenge,

three fish form each group were random

selected for estimated the number of

parasites as described previously by

Osman et al. (2009). The parasites

infection level was assessed by scoring,

no infection = 0, <50 trophonts fish-1

=

1, 50-100 trophonts fish-1

=2 and >100

trophonts fish-1

=3. One week after

challenge, the survival rate of fish in

each tank was determined. The

experiment was done in duplicate.

Statistical analysis

The data was analyzed using SPSS for

Windows Version 17.0. Parameter

values were reported as mean±standard

deviation. The differences in parameter

values of each treatment were examined

using one-way ANOVA with Scheffé

post hoc tests (p<0.05).

Results

Anti-Ich antibody titer

Anti-Ich antibody was not detected in

pre-immunized fish whereas it was

detected in fish serum that was sampled

from treatments 3-8 (Table 1). Non-

immunized fish and fish immunized

with BSA did not produce antibody

against Ich (Table 1). Fish immunized

with live trophonts revealed higher

antibody titers against Ich than those

immunized with sonicated trophonts.

Fish immunized with live trophonts at a

protein concentration 65 µg g-1

fish

showed the highest antibody titer

against Ich at a dilution 1:1,000

(p<0.05). These fish had significantly

higher anti-Ich antibody titers than fish

immunized with sonicated trophonts at

the same protein concentration.

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Moreover, they had significantly higher

antibody titer against Ich than fish

immunized with live or sonicated

trophonts at 45 and 55 µg protein g-1

fish. Fish immunized with sonicated

trophonts at 65 µg protein g-1

fish

showed antibody titers at a 1:100

dilution similar to fish immunized with

live trophonts at 45 and 55 µg protein g-

1 fish. Fish immunized with sonicated

trophonts at 45 and 55 µg protein g-1

fish showed antibody titers against Ich

at a 1:10 dilution. However, no

statistical difference was found between

antibody titers from fish immunized

with live trophonts at 45 and 55 µg

protein g-1

fish including fish

immunized with sonicated trophonts at

45 and 55 µg protein g-1

fish.

Table 1: ELISA absorbance at 405 nm of non-immunized and immunized Basa fish (Pangasius

bocourti) serum 21 days post immunization.

Treatment Dilution A405nm

Pre-immunized 1:10 0.000±0.000a*

Non-immunized 1:10 0.032±0.005a

Immunized with BSA at 65 µg g-1

fish 1:10 0.031±0.006a

Immunized with live trophonts at 45 µg g-1

fish

1:10 0.302±0.032b

1:102 0.147±0.016

c

1:103 0.043±0.008

a

1:104 0.009±0.006

a

1:105 0.000±0.000

a


fish

1:10 0.334±0.077b

1:102 0.196±0.045

c

1:103 0.077±0.019

a

1:104 0.017±0.005

a

1:105 0.000±0.000

a


fish

1:10 0.369±0.060b

1:102 0.201±0.051

b

1:103 0.114±0.015

c

1:104 0.033±0.002

a

1:105 0.000±0.000

a

Immunized with sonicated trophonts at 45 µg g-1

fish

1:10 0.183±0.013c

1:102 0.0490.010

a

1:103 0.0060.003

a

1:104 0.0000.000

a

1:105 0.0000.000

a


fish

1:10 0.218±0.008c

1:102 0.0810.010

a

1:103 0.0240.004

a

1:104 0.0050.004

a

1:105 0.0000.000

a


fish

1:10 0.2560.038b

1:102 0.173±0.021

c

1:103 0.0920.016

a

1:104 0.0060.004

a

1:105 0.0000.000

a

*Different superscripts indicate statistically significant differences.

The results determined using western

immunoblotting were similar to ELISA.

Anti-Ich antibody was not detected in

pre-immunized fish, non-immunized

fish and fish immunized with BSA.

Fish immunized with live or sonicated

trophonts showed specific protein bands

at approximately 66 kDa and below 14

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kDa (Fig.1A and 1B). Fish immunized

with live and sonicated trophonts at a

concentration 65 µg protein g-1

fish

indicated the highest titer, 1:100. Both

of fish immunized with live and

sonicated trophonts at a concentration

45 and 55 µg protein g-1

fish revealed a

titer at a 1:10 dilution. However,

protein band intensity of fish

immunized with live trophonts was

higher than that of fish immunized with

sonicated trophonts.

A

B

Figure 1: Western immunoblotting of immunized Basa fish (Pangasius bocourti) serum 21 days

post immunization. (A) Fish immunized with live trophonts, lane M: marker, lane 1: pre-

immunized fish at dilution 1:10, lanes 2 and 3: immunized with 45 µg protein g-1

fish at

dilution 1:10 and 1:100, lanes 4 and 5: immunized with 55 µg protein g-1

fish at dilution

1:10 and 1:100 and lanes 6 and 7: immunized with 65 µg protein/g fish at dilution 1:10

and 1:100. (B) fish immunized with sonicated trophonts lane M: marker, lane 1: non-

immunized serum at dilution 1:10, lane 2 immunized with BSA 65 µg protein g-1

fish,

lanes 3 and 4: immunized with 45 µg protein g-1

fish at dilutions 1:10 and 1:100, lanes 5

and 6: immunized with 55 µg protein g-1

fish at dilution 1:10 and 1:100 and lanes 7 and

8: immunized with 65 µg protein g-1

fish at dilution 1:10 and 1:100.

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Survival rate of fish with Ich

After challenge with Ich for two days,

the number of parasites on the fish was

estimated. The results showed that Non-

immunized fish and fish immunized

with BSA had similar higher parasites

infection level per fish than fish

immunized with live and sonicated

trophonts. Fish immunized with live

trophonts at a level of 65 µg g-1

fish had

the lowest number of parasites per fish

(Table 2). Non-immunized fish and fish

immunized with BSA showed heavy

infection, whereas fish immunized with

live or sonicated trophonts revealed

light infection. First dead fish in Non-

immunized group was found three days

post challenge while fish immunized

with live trophonts was found dead four

days post challenge. After challenge

with Ich for one week, Non-immunized

fish showed the highest mortality,

100% (Table 2). All non-immunized

fish has been found dead four days after

challenge. Fish immunized with BSA

had a low survival rate of 3.33%

(Table 2). The survival rate of fish

immunized with live trophonts at a

level of 65 µg g-1

fish had the highest

and most statistically significant

difference (Table 2) (p<0.05). There

were no statistically significant

differences between fish immunized

with live and sonicated trophonts at

lower protein concentrations. Two

weeks post challenge, all survive fish

that immunized with live and sonicated

trophonts at a level of 45, 55 and 65 µg

g-1

fish were still survive while all

survive fish that immunized with BSA

was mortal (data not shown).

Table 2: Parasites infection level per fish two days post challenge and survival rate one week post

challenge of non-immunized and immunized Basa fish (Pangasius bocourti).

Treatment parasites infection

level Survival rate (%)

Non-immunized 3 0.000.00a*

Immunized with BSA at 65 µg g-1

fish 3 3.335.77a


fish 2 33.335.77b


fish 2 40.0010.00b


fish 1 63.335.77c


fish 2 30.0010.00b


fish 1 33.3315.28b


fish 1 43.335.77b

*Different superscripts indicate the statistical significant differences.

Discussion

The anti-Ich antibody titers observed in

this study were similar to other fish

immunization using the same method of

antigen injection (Dalgaard et al., 2002;

Xu et al., 2004; Xu et al., 2008a; Xu et

al., 2008b; Osman et al., 2009; Martins

et al., 2011). Intraperitoneal injection

was potential method for fish

immunization (Burk et al., 1990).

Non-immunized fish and fish

immunized with BSA did not produce

anti-Ich antibody according to a

previous report (Xu and Klesius, 2013).

Fish immunized with live trophonts

showed higher antibody titers than that

of fish immunized with sonicated

trophonts. These results indicated that

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live trophonts exhibited better

antigenicity than sonicated trophonts.

Therefore, fish immunized with live

trophonts produced significantly higher

antibody titers than fish immunized

with inactivated trophonts. These

results are in accordance with previous

studies of immunization with

inactivated antigens as formalin killed

and freeze-thawed trophonts that

reported lower antibody production

than when immunized with live

antigens (Xu et al., 2008a, b). However,

live trophonts that used as antigen has

the advantage when compare with live

theronts that used as antigen in the

previous study because antigen

preparation is less complicated.

Moreover, the antigen dose had an

effect on antibody production. The

antibody of fish immunized with 65 µg

of live and sonicated trophont protein g-

1 fish revealed significantly higher

antibody than that of fish immunized

with 45 and 55 µg trophont protein g-1

fish. However, no statistical difference

of antibody titer was found in fish

immunized with live and sonicated

trophonts at concentrations of 45 and 55

µg g-1

fish. These results agree with a

previous study of antibody production

for immune protection, which found

that a suitable concentration of antigen

was required for immune protection

(Xu et al., 2008b). In previous studies,

fish immunoglobulin (Ig) as IgM and

IgD were produced in immunized

channel catfish, while IgM and IgT

were detected in rainbow trout (Olsen et

al., 2011; Heinecke and Buchman,

2013; Xu et al., 2016). From this, we

hypothesized that IgM should be

produced in Basa fish as a serum

antibody. Moreover, another immune

response against Ich, increased white

blood cell counts, was also found in fish

(Abdel-Hafez et al., 2014; Tancredo et

al., 2015). We suggest a study of

immunological parameter responses as

a future study. Furthermore, multiple

serotypes immobilization antigen of Ich

are found and have been classified into

five serotypes referred to serotypes A,

B, C, D and E (Dickerson and Clark,

1996). There are the differences in

virulence and protein molecular weight

between serotypes. For example,

serotypes A infected fish at a lower

level than serotypes D but all infected

fish with serotypes A died. In the

previous study, cross-reactivity of anti-

Ich antibody with different serotypes of

immobilization antigen was found

(Swennes et al., 2007). Probably, anti-

Ich antibody from immunized fish with

trophonts in this study may be reacting

with multiple serotypes immobilization

antigen.

The results obtained from western

immunoblotting revealed that fish

antibody could react with Ich antigen

protein at approximately 66 kDa and

below 14 kDa. In contrast with a

previous report, a protein

approximately 55 kDa acting as a

surface immobilization antigen of Ich

was not found in this research (Wang

and Dickerson, 2002; Maki and

Dickerson, 2003). Moreover, proteins at

approximately 34, 39, 45 and 46 kDa

acting as antigens against Ich

membrane proteins play an important

role in fish immunization (Wang et al.,

2002). These proteins were not found in

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the current study. We theorize that the

proteins at approximately 66 kDa and

below 14 kDa should be important Ich

antigens in Basa fish. We also

hypothesize that the proteins at

approximately 66 kDa may promote a

surface immobilization antigen against

Ich. However, the proteins below 14

kDa should be the interaction of

antibody with the reduced parts of

proteins as break down products from

the immobilization antigens (i-antigens;

ranging in size from 40 to 70 kDa) (Lin

et al., 1996; Swennes et al., 2006).

In this study, non-immunized fish

and fish immunized with BSA showed

heavy infection and died after few days.

This was similar to results of a study of

infected Nile Tilapia that exhibited a

mean of five days to death (Xu et al.,

2014). The immunized fish could

produce protective antibody against Ich.

They had high anti-Ich antibody titers

and exhibited light infections with high

survival rates. Fish immunized with

high level of antigen had the lowest

number of parasites infection per fish

that exhibit the quantitative exact result

of protection. The results showed that

the immune response and immune

protection were proportional to anti-Ich

antibody. However, the fish were not

fully protected and this may be due to

an in-sufficient level of anti-Ich

antibodies or other factors, as was

observed in other studies (Xu et al.,

2008a; Osman et al., 2009). We

proposed that Ich successfully penetrate

the epithelium of immune fish but are

forced to exit due to cutaneous antibody

attachment.

In conclusion, Basa fish immunized

with live and sonicated trophonts by IP

injection showed an immune response

against Ich. Basa fish immunized with

live trophonts produced higher anti-Ich

antibody titers and had greater survival

rates than those of fish immunized with

sonicated trophonts. Therefore,

immunization of Basa fish with live and

sonicated Ich by IP injection enhance

protection against this parasite.

Acknowledgement

This research was supported by

National Research Council of Thailand

(NRCT). The authors wish to thank

Siwatch Sakulwongworachote for

excellent support in maintenance of the

Basa fish. The authors also thank

Uraiwan Piasoongnoen, Nakhon

Phanom Inland Fisheries Research and

Development Center, for parasite

collection.

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